Adhesion between Phosphatidylethanolamine Bilayers - Langmuir

Mar 20, 1996 - The specific problem concerns the hydration properties of bilayers of the membrane lipid phosphatidylethanolamine (PE), which imbibe mu...
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Adhesion between Phosphatidylethanolamine Bilayers Thomas J. McIntosh*,† and Sidney A. Simon‡ Departments of Cell Biology, Neurobiology, and Anesthesiology, Duke University Medical Center, Durham, North Carolina 27710 Received October 5, 1995. In Final Form: December 7, 1995X The goal of this study is to provide additional information on the short-range interactions that determine the adhesion energy between bilayer surfaces. The specific problem concerns the hydration properties of bilayers of the membrane lipid phosphatidylethanolamine (PE), which imbibe much less water than bilayers composed of the other common ziwtterionic membrane lipid, phosphatidylcholine (PC). The osmotic stress/ X-ray diffraction method was used to measure pressure-distance relations for PE and PC bilayers containing known mole fractions of the charged lipid phosphatidic acid (PA). The addition of 20 mol % PA to either PC or PE bilayers swelled the bilayers by an amount predictable from electrostatic double-layer theory. However, whereas the addition of 5 mol % PA disjoined PC bilayers, it did not change the fluid space between PE bilayers. By calculating the magnitude of the electrostatic pressure necessary to disjoin the bilayers, we estimate the adhesion energy for gel phase PE bilayers to be about -0.7 erg/cm2, a value considerably larger than previously measured values for gel phase PC bilayers. The magnitude of the adhesion energy indicates that, in addition to the attractive van der Waals pressure, there is another attractive pressure between adjacent PE layers that prevents them from swelling to the extent of PC bilayers. We argue that a small fraction of direct electrostatic interbilayer interactions or indirect hydrogenbonded water interactions between the N+H3 group in one bilayer and the PO4- group in the apposing bilayers could account for the additional attractive interactions in PE bilayers.

Introduction Two of the most common phospholipids in biological membranes are the uncharged lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Extensive studies have been performed on the structural,1-7 polymorphic,8-14 dynamic,15,16 and thermal14-20 properties of each of these phospholipids. One major difference between these lipid classes is that bilayers composed of PC imbibe significantly more water than do PE bilayers,21-25 so that in both the gel and liquid crystalline multilamellar phases the distance between apposing * Corresponding author, e-mail address: tom mcintosh@cellbio. duke.edu. † Department of Cell Biology. ‡ Department of Neurobiology and Anesthesiology. X Abstract published in Advance ACS Abstracts, March 1, 1996. (1) Levine, Y. K.; Wilkins, M. H. F. Nature New Biol. 1971, 230, 69. (2) Lesslauer, W.; Cain, J. E.; Blasie, J. K. Proc. Nat. Acad. Sci. USA 1972, 69, 1499. (3) Hitchcock, P. B.; Mason, R.; Thomas, K. M.; Shipley, G. G. Proc. Nat. Acad. USA 1974, 71, 3036. (4) Torbet, J.; Wilkins, M. H. F. J. Mol. Biol. 1976, 62, 447. (5) McIntosh, T. J. Biophys. J. 1980, 29, 237. (6) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21. (7) McIntosh, T. J.; Simon, S. A. Biochemistry 1986, 25, 4948. (8) Luzzati, V.; Gulik-Krzywicki, T.; Tardieu, A. Nature 1968, 218, 1031. (9) Tardieu, A.; Luzzati, V.; Reman, F. C. J. Mol. Biol. 1973, 75, 711. (10) Cullis, P. R.; DeKruijff, B. Biochim. Biophys. Acta 1978, 513, 31. (11) Seddon, J. M.; Harlos, K.; Marsh, D. J. Biol. Chem. 1983, 258, 3850. (12) Seddon, J. M.; Cevc, G.; Kaye, R. D.; Marsh, D. Biochemistry 1984, 23, 2634. (13) Gruner, S. M.; Tate, M. W.; Kirk, G. L.; So, P. T. C.; Turner, D. C.; Keane, D. T.; Tilcock, C. P. S.; Cullis, P. R. Biochemistry 1988, 27, 2853. (14) Lewis, R. N. A. H.; McElhaney, R. N. Biophys. J. 1993, 64, 1081. (15) Seelig, J.; Seelig, A. Q. Rev. Biophys. 1980, 13, 19. (16) Blume, A. In Phospholipids Handbook; Cevc, G., Ed.; Marcel Dekker, Inc.: New York, 1993. (17) Mabrey, S.; Sturtevant, J. M. Proc. Nat. Acad. Sci. USA 1976, 73, 3862. (18) Chen, S. C.; Sturtevant, J. M.; Gaffney, B. J. Proc. Natl. Acad. Sci. USA 1980, 77, 5060. (19) Wilkinson, D. A.; Nagle, J. F. Biochemistry 1979, 18, 4244. (20) Wilkinson, D. A.; Nagle, J. F. Biochemistry 1981, 20, 187. (21) Jendrasiak, G. L.; Mendible, J. C. Biochim. Biophys. Acta 1976, 424, 149.

bilayers is considerably larger for PC than for PE bilayers.7,24,26-28 This observation of a smaller fluid space for PE bilayers has implications in terms of the interactions between bilayers, as it means that PE bilayers have either weaker interbilayer repulsive pressure(s) or stronger interbilayer attractive pressure(s) compared to those of PC bilayers. Several nonspecific interactions between uncharged phospholipid bilayers have been either proposed theoretically or demonstrated experimentally, including the attractive van der Waals pressure,29,30 the repulsive hydration pressure,25,31,32 and repulsive steric pressures due to headgroup movement,33,34 molecular protrusions,35,36 and bilayer undulations.37,38 There have been several proposals to explain the smaller fluid spacing for PE bilayers. For example, compared to PC bilayers, it has been argued that PE bilayers have smaller repulsive hydration pressure,13 stronger van der Waals attractive pressure,26 less (22) Cevc, G.; Marsh, D. Phospholipid bilayers. Physical Principles and Models; John Wiley & Sons: New York, 1987. (23) Nagle, J. F.; Wiener, M. C. Biochim. Biophys. Acta 1988, 942, 1. (24) Rand, R. P.; Fuller, N.; Parsegian, V. A.; Rau, D. C. Biochemistry 1988, 27, 7711. (25) Rand, R. P.; Parsegian, V. A. Biochim. Biophys. Acta 1989, 988, 351. (26) Lis, L. J.; McAlister, M.; Fuller, N.; Rand, R. P.; Parsegian, V. A. Biophys. J. 1982, 37, 657. (27) Marra, J.; Israelachvili, J. Biochemistry 1985, 24, 4608. (28) McIntosh, T. J.; Simon, S. A. Ann. Rev. Biophys. Biomol. Struct. 1994, 23, 27. (29) Parsegian, V. A.; Nimham, B. W. Nature 1969, 224, 1197. (30) Mahanty, J.; Nimham, B. W. Dispersion Forces; Academic Press: New York, 1976. (31) Marcelja, S.; Radic, N. Chem. Phys. Lett. 1976, 42, 129. (32) LeNeveu, D. M.; Rand, R. P.; Parsegian, V. A.; Gingell, D. Biophys. J. 1977, 18, 209. (33) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1987, 26, 7325. (34) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1989, 28, 17. (35) Israelachvili, J. N.; Wennerstrom, H. Langmuir 1990, 6, 873. (36) Israelachvili, J. N.; Wennerstrom, H. J. Phys. Chem. 1992, 96, 520. (37) Helfrich, W. Z. Naturforsch. 1973, 28C, 693. (38) Evans, E. A.; Parsegian, V. A. Proc. Nat. Acad. Sci. USA 1986, 83, 7132.

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dynamical freedom in the PE headgroup due to intrabilayer hydrogen bonds,39 large attractive hydration pressure,24 H-bonding water bridges between PE bilayers,7,40 and direct electrostatic interactions between the amine and phosphate groups on apposing bilayers.7,39 Experimentally it is difficult to distinguish among these various possibilities, in part due to problems in extracting the attractive and repulsive pressures from measured pressure-distance relations because of the extremely small distances between adjacent PE bilayers.7,27,28 In this paper we use the osmotic stress/X-ray diffraction method32,41,42 to measure pressure-distance relations between PE and PC bilayers over an expanded distance regime. The interbilayer distance is extended by incorporating into both PE and PC bilayers various concentrations of the negatively charged lipid phosphatidic acid (PA), which adds a known electrostatic repulsive pressure to the bilayer surface.43 The experiments are designed to (1) provide an estimate of the adhesion energy between gel phase PE bilayers and (2) test the hypothesis that there is an additional attractive interaction that operates between PE but not PC bilayers. The rationale is that if an additional attractive interaction exists, then it should take a larger repulsive electrostatic pressure (the addition of more negatively charged lipid) to swell PE bilayers than PC bilayers. In strong support of this idea is the previous work of Van der Kleij et al.,44 who found that the lamellar repeat period of PE/PA mixtures remains virtually constant for 0-4 mol % PA whereas the repeat period of PC bilayers increases significantly with the incorporation of 4 mol % PA. Here we measure complete pressuredistance relations and use these data to determine the energy necessary to disjoin PE bilayers. To simplify the analysis, we use gel phase bilayers, where the protrusion and undulation pressures are negligible.28,45,46 Materials and Methods Dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), and dipalmitoylphosphatidic acid, sodium salt (DPPA) were obtained from Avanti Polar Lipids (Alabaster, AL). Dextran of average molecular weight 580,000 and polyvinylpyrrolidone (PVP) of average molecular weight 40,000 were purchased from Sigma Chemical Company (St. Louis, MO). Dextran and PVP solutions were made in a buffer containing 100 mM NaCl and 20 mM HEPES at pH 7. X-ray diffraction analysis was performed on suspensions of multiwalled vesicles composed of various mixtures of DPPC: DPPA and DPPE:DPPA. Osmotic stress was applied to the liposomes by incubating them in aqueous solutions of dextran or PVP. Since these polymers are too large to enter the lipid lattice, they compete for water with the lipid multilayers, thereby applying an osmotic pressure.32,41 Osmotic pressures for dextran and PVP solutions have been published.47 Lipid suspensions were made by first codissolving in chloroform appropriate DPPE: DPPA and DPPC:DPPA mixtures. The chloroform was removed by rotary evaporation, and multilamellar suspensions were prepared by adding excess polymer solution (10 mg of lipid per mL of polymer solution) and incubating with extensive vortexing above the lipid’s phase transition temperature. To ensure equilibration of the NaCl across the multilayers, several freezethaw cycles were used. The suspensions were pelleted with a (39) Damodaran, K. V.; Merz, K. M., Jr. Biophys. J. 1994, 66, 1076. (40) Boggs, J. M. Biochim. Biophys. Acta 1987, 906, 353. (41) Parsegian, V. A.; Fuller, N.; Rand, R. P. Proc. Nat. Acad. Sci. USA 1979, 76, 2750. (42) McIntosh, T. J.; Simon, S. A. Biochemistry 1986, 25, 4058. (43) Evans, E.; Needham, D. J. Phys. Chem. 1987, 91, 4219. (44) van der Kleij, A. A. M.; Oltmans, F.; Geurts van Kessel, W. S. M.; De Kruijff, B. Chem. Phys. Lipids 1988, 47, 123. (45) McIntosh, T. J.; Simon, S. A. Biochemistry 1993, 32, 8374. (46) Konig, S.; Bayerl, T. M.; Coddens, G.; Richter, D.; Sackmann, E. Biophys. J. 1995, 68, 1871. (47) Parsegian, V. A.; Rand, R. P.; Fuller, N. L.; Rau, R. C. Methods Enzymol. 1986, 127, 400.

Langmuir, Vol. 12, No. 6, 1996 1623 bench centrifuge, sealed in quartz glass X-ray capillary tubes, and mounted in a point collimation X-ray camera. X-ray diffraction patterns were recorded on stacks of Kodak DEF X-ray film which were densitometered with a Joyce-Loebl microdensitometer.33,42,48 After background subtraction, integrated intensities, I(h), were obtained for each order h by measuring the area under each diffraction peak. For these patterns from unoriented suspensions49,50 the structure amplitudes F(h) were set equal to {h2I(h)}1/2. Electron density profiles, F(x), on a relative electron density scale were calculated from

∑exp{iφ(h)}F(h) cos(2πxh/d)

F(x) ) (2/d)

(1)

where x is the distance from the center of the bilayer, d is the lamellar repeat period, φ(h) is the phase angle for order h, and the sum is over h. Phase angles were determined by the use of the sampling theorem51 as described in detail previously.34,42,52 Electron density profiles are at a resolution of d/2hmax ≈ 7 Å.

Results The X-ray diffraction patterns of suspensions of DPPE, 19:1 DPPE:DPPA, 4:1 DPPE:DPPA, DPPC, 19:1 DPPC: DPPA, and 4:1 DPPC:DPPA consisted of several low-angle reflections that indexed as orders of a single lamellar repeat period and one or two sharp wide-angle reflections. For suspensions of 9:1 DPPE:DPPA, two lamellar repeat periods were observed at low applied pressures (see below). For all suspensions of DPPE or DPPE:DPPA the wideangle patterns consisted of a single sharp reflection at 4.13 Å. Such sharp wide-angle patterns are typical of multibilayers in the Lβ gel phase, where the lipid hydrocarbon chains are untilted or nearly perpendicular to the plane of the bilayer.5,9 For all suspensions containing DPPC or DPPC:DPPA the wide-angle patterns contained a sharp reflection at 4.21 Å and a broad reflection centered at 4.10 Å. These patterns are typical of bilayers in the Lβ′ phase, where the hydrocarbon chains are tilted with respect to the plane of the bilayer.5,9 For all specimens of DPPE:DPPA or DPPC:DPPA bilayers, the wide-angle patterns were invariant over the range of applied pressures used in these experiments. The lamellar repeat period (d) depended strongly on both the bilayer composition and the applied osmotic pressure (P). Figure 1A shows a plot of the logarithm of applied pressure (log P) versus the lamellar repeat period for suspensions of DPPE:DPPA. For DPPE bilayers, the lamellar repeat period decreased from 62.5 Å at zero applied pressure (denoted by arrow in Figure 1A) to 60.2 Å at log P ) 7.4. The pressure distance relationships were, within experimental error, identical for DPPE and 19:1 DPPE:DPPA (Figure 1A). For 9:1 DPPE:DPPA for values of log P < 6, two lamellar repeat periods were observed; one repeat period was the same as that observed for DPPE (or 19:1 DPPE:DPPA), whereas the other value of d was significantly larger (Figure 1A). The relative intensities were considerably larger for the orders of the smaller repeat period, indicating that most of the specimen was in the low periodicity phase observed for DPPE. For 9:1 DPPE:DPPA and values of log P > 6, a single repeat period was observed, very similar to that observed for DPPE or 19:1 DPPE:DPPA. For 4:1 DPPE:DPPA and log P < 7, the lamellar repeat period was considerably larger (48) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biochemistry 1989, 28, 7904. (49) Blaurock, A. E.; Worthington, C. R. Biophys. J. 1966, 6, 305. (50) Herbette, L.; Marquardt, J.; Scarpa, A.; Blasie, J. K. Biophys. J. 1977, 20, 245. (51) Shannon, C. E. Proc. Inst. Radio Eng. NY 1949, 37, 10. (52) McIntosh, T. J.; Holloway, P. W. Biochemistry 1987, 26, 1783.

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Figure 1. The logarithm of applied pressure (log P) plotted versus the lamellar repeat period (d) for (A) DPPE (open circles), 19:1 DPPE:DPPA (open squares), 9:1 DPPE:DPPA (open triangles), and 4:1 DPPE:DPPA (open diamonds) and for (B) DPPC (solid circles), 19:1 DPPC:DPPA (solid squares), and 4:1 DPPE:DPPA (solid diamonds). For log P < 6, two lamellar phases were observed for 9:1 DPPE:DPPA specimens. Arrows in (A) and (B) denote the repeat periods in excess water (no applied pressure) for DPPE and DPPC, respectively. Values for DPPC were taken from McIntosh and Simon.42

than that observed for DPPE, wheras for log P > 7 the observed repeat periods were nearly identical to those of DPPE. Figure 1B shows a plot of log P versus d for suspensions of DPPC:DPPA. For DPPC bilayers, the lamellar repeat period decreased with increasing pressure from a value of 63.6 Å in excess buffer with no applied pressure (denoted by arrow) to a value of 57.8 Å at log P ) 7.8.42 For 19:1 DPPC:DPPA for log P < 6 and for 4:1 DPPC:DPPA for log P < 7, the lamellar repeat period was larger than that for DPPC (Figure 1B). However, for log P > 7, the log P versus d plots were similar to those for DPPC, 19:1 DPPC: DPPA, and 4:1 DPPC:DPPA. The lamellar repeat period contains the widths of both the lipid bilayer and the fluid space between adjacent bilayers. To obtain information on the structure of the bilayer and width of the interbilayer fluid spaces for both DPPE:DPPA and DPPC:DPPA suspensions, a Fourier analysis of the diffraction data was performed. Figure

2A,B shows plots of the observed structure amplitudes for suspensions of DPPE:DPPA and DPPC:DPPA, respectively, for the repeat periods shown in Figure 1. The solid lines correspond to the continuous Fourier transforms of DPPE (Figure 2A) and DPPC (Figure 2B) calculated using the sampling theorem51,53,54 and the phase angles previously determined for these lipids.5,7,42 The structure amplitude at the origin corresponds to the value calculated for DPPC by Wiener et al.55 Three major points can be made from the data in Figure 2A,B. First, although the transforms of DPPE and DPPC both had three peaks in the region of reciprocal spacing 0.01-0.08 Å-1, there were distinct differences in the transforms, indicating differences in the bilayer structure. In particular, the corresponding peaks and nodes were at larger values of (53) Sayre, D. Acta Crystallogr., Sect. B 1952, 5, 843. (54) Worthington, C. R. J. Appl. Cryst. 1988, 21, 322. (55) Wiener, M. C.; Suter, R. M.; Nagle, J. F. Biophys. J. 1989, 55, 315.

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Figure 2. Measured structure amplitudes for (A) DPPE (open circles), 9:1 DPPE:DPPA (open triangles), and 4:1 DPPE:DPPA (open diamonds) and (B) DPPC (solid circles), 19:1 DPPC:DPPA (solid squares), and 4:1 DPPE:DPPA (solid diamonds). Values for DPPC were taken from McIntosh and Simon.42 The solid lines represent the continuous Fourier transforms of a single bilayer of (A) DPPE and (B) DPPC calculated by use of the sampling theorem (see text for details).

reciprocal space for DPPE than for DPPC. Second, the structure amplitudes of 19:1 DPPE:DPPA and 4:1 DPPE: DPPA fell on the same transform as DPPE (Figure 2A), and the structure amplitudes of 19:1 DPPC:DPPA and 4:1 DPPC:DPPA fell on the same transform as DPPC (Figure 2A). This indicates that the addition of DPPA did not significantly change the structure of the matrix bilayer. Third, in the cases of both DPPE:DPPA and DPPC:DPPA suspensions, the structure amplitudes obtained at all values of applied pressures fell on the continuous transforms (solid lines). This implies that for both DPPE:DPPA and DPPC:DPPA suspensions the bilayer structure remained nearly constant over the range of applied osmotic pressures. Figure 3A,B shows electron density profiles for DPPE: DPPA and DPPC:DPPA bilayers, respectively, obtained at similar values of applied pressure. For each bilayer system, the high density peaks (near (24 Å for DPPE: DPPA and near (21 Å for DPPC:DPPA) correspond to the high density phospholipid headgroups and the troughs at

0 Å correspond to the localization of the low electron density terminal methyl groups in the bilayer center. The medium density regions between the terminal methyl troughs and the headgroup peaks correspond to the lipid methylene chains, and the medium density regions at the outer edges of the profiles correspond to the fluid layers between adjacent bilayers. These profiles show several structural features. First, the width of the bilayer, as measured by the peak-to-peak separation (dpp) in the profiles, was larger for DPPE than for DPPC. For DPPE the peak-to-peak separation was 47.3 ( 0.3 Å (mean ( standard deviation for n ) 4 experiments), whereas for DPPC dpp was observed to be 41.9 ( 0.6 Å (n ) 11 experiments).42 Second, for DPPE the addition of 5 or 20 mol % DPPA did not markedly change the electron density profiles or the values of dpp, as dpp was 47.0 ( 0.6 Å (n ) 2) for 19:1 DPPE:DPPA and 47.5 ( 1.1 Å (n ) 4) for 4:1 DPPE:DPPA. For DPPC, the electron density profile did not markedly change with the addition of DPPA, although the measured values of dpp increased slightly to 42.8 ( 1.0

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Figure 3. Electron density profiles of (A) DPPE, 9:1 DPPE: DPPA, and 4:1 DPPE:DPPA and (B) DPPC, 19:1 DPPC:DPPA, and 4:1 DPPE:DPPA. All profiles are from samples with applied pressures of 7.4 < log P < 7.5.

Å (n ) 4) for 19:1 DPPC:DPPA and 43.7 ( 1.0 Å (n ) 5) for 4:1 DPPC:DPPA. No systematic change in dpp was observed with increasing applied pressure for any of these bilayer systems. Figure 4A,B shows log P plotted versus d-dpp for DPPE: DPPA and DPPC:DPPA suspensions, respectively. The parameter d-dpp, which represents the distance between headgroup peaks from apposing bilayers and includes both the fluid space between adjacent bilayers and the outer portion of the lipid headgroup region of each bilayer, is a convenient parameter to use in these studies since the headgroup peak location is near the phosphate group and thus provides a good reference point for calculations of electrostatic repulsive pressures (see below). Also, as we have done previously,33,42 we use the electron density profiles and crystal structures of PE3 and PC56 to estimate the physical edge of the PE and PC bilayer, which are indicated by the vertical-dotted lines in Figure 4A,B, respectively. That is, as it has been shown for both PC2 and PE3 bilayers, at this resolution the headgroup peaks in electron density profiles fall between the phosphate moiety and the glycerol backbone, or about 4 Å from the physical edge of the PE bilayer and about 5 Å from the physical edge of the PC bilayer.42 Thus, these dotted lines represent the physical edge of the bilayer, assuming that the lipid headgroups have the same conformations as found in crystals. We note that our estimate for the bilayer (56) Pearson, R. H.; Pascher, I. Nature 1979, 281, 499.

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thickness of DPPE bilayers (55.1 Å) is in excellent agreement with the observed repeat period of 55.5 Å for completely dehydrated DPPE bilayers.57 The pressure-distance relations for DPPE:DPPA (Figure 4A) displayed the following features. First, the log P versus d-dpp relations were almost identical for DPPE and 19:1 DPPE:DPPA for all applied pressures, with d-dpp having a maximum value of about 15 Å at low applied pressures and decreasing to about 13 Å at high applied pressures (log P > 7.5). Second, for 4:1 DPPE: DPPA the values of d-dpp were significantly larger than those of DPPE or 19:1 DPPE:DPPA for log P < 6.5. However, for 4:1 DPPE:DPPA there was a sharp upward break in the pressure-distance data at log P ≈ 7, so that the data points for DPPE, 19:1 DPPE:DPPA, and 4:1 DPPE:DPPA nearly superimposed for log P > 7. Such upward breaks have been observed in other charged lipid bilayers.58-60 The pressure-distance relations for DPPC: DPPA (Figure 4B) showed that DPPC had a maximum value of d-dpp ≈ 22 Å at log P < 4 and monotonically decreased to d-dpp ≈ 16 Å for log P ) 7.5. Both 19:1 and 4:1 DPPC:DPPA bilayers had larger fluid spaces than DPPC at small applied pressures. For 19:1 DPPC:DPPA the values of d-dpp were larger than those of DPPC for log P < 5.5 but were similar to those of DPPC for log P > 5.5. For 4:1 DPPC:DPPA the bilayers swelled for log P < 7 but had values similar to those of DPPC for log P > 7. There were three major differences between the DPPE: DPPA (Figure 4A) and DPPC:DPPA (Figure 4B) pressuredistance relations. First, 19:1 DPPC:DPPA bilayers swelled apart (disjoined) at low applied pressures, whereas 19:1 DPPE:DPPA bilayers did not. Second, the pressuredistance relation was shifted to smaller values of d-dpp for the DPPE bilayers; the pressure-distance relations for DPPE and DPPC were offset from each other by about 7 Å at low applied pressures and by about 4 Å at high pressures (also see Figure 5). Third, the distance between the physical edge of the bilayer (dotted lines) and the values of d-dpp was larger, particularly at small applied pressures, for DPPC than for DPPE. This demonstrates that the fluid space was greater between PC bilayers than between PE bilayers, in agreement with previous observations.7,24,26,27 That is, as the applied pressure was increased from zero to 5 × 107 dyn/cm2, the fluid space between adjacent DPPE bilayers (as measured from the edge of the bilayer) decreased from 7.4 to 5.1 Å whereas the fluid space between adjacent DPPC bilayers decreased from 11.7 to 5.9 Å. Thus, the overall picture that reflects the differences between DPPE and DPPC can be appreciated by noting that over the entire range of osmotic pressures the fluid space decreased about 2.3 and 5.8 Å for DPPE and DPPC, respectively (Figure 4A,B). Since the area/molecule5,9 for DPPE is 40 Å2 and for DPPC is 48 Å2, it follows that over this range of applied pressures water volumes of 92 and 278 Å3 were removed from between apposing lipid molecules in DPPE and DPPC multilayers, respectively. This implies that, per pair of apposing lipid molecules, about three water molecules were removed from between DPPE bilayers, whereas about nine water molecules were removed from between DPPC bilayers, and demonstrates that DPPC imbibes significantly more water than does DPPE. (57) Suwalsky, M.; Knight, E. Z. Naturforsch. 1982, 37c, 1157. (58) Cowley, A. C.; Fuller, N. L.; Rand, R. P.; Parsegian, V. A. Biochemistry 1978, 17, 3163. (59) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biophys. J. 1990, 57, 1187. (60) Parsegian, V. A.; Rand, R. P.; Fuller, N. L. J. Phys. Chem. 1991, 95, 4777.

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Figure 4. The logarithm of applied pressure (log P) plotted versus the lamellar repeat period (d) minus the peak-to-peak distance in the electron density profiles (dpp) for (A) DPPE (open circles), 19:1 DPPE:DPPA (open squares), and 4:1 DPPE:DPPA (open diamonds) and (B) DPPC (solid circles), 19:1 DPPC:DPPA (solid squares), and 4:1 DPPE:DPPA (solid diamonds). In panels (A) and (B), the dotted lines denote the approximate position of the edge of the bilayer and the arrows denote the values of d-dpp at zero applied pressure. Values for DPPC were taken from McIntosh and Simon.42

Discussion The results presented above provide information on the effects of charged lipids on the structure of PE and PC bilayers and on the interactions between both PE and PC bilayers. Bilayer Structure. The wide-angle diffraction data, the reciprocal space analysis of the structure amplitudes (Figure 2), and the electron density profiles (Figure 3) all show that the incorporation of 5-20 mol % DPPA did not appreciably change the packing of the acyl chains in either DPPE or DPPC bilayers. The only slight structural modification was a small increase in the values of dpp with the incorporation of DPPA into DPPC bilayers. This likely arises from a small decrease in the hydrocarbon chain tilt. The bilayer thickness, as indicated by the measured values of dpp, was larger for DPPE or DPPE:DPPA bilayers than for DPPC or DPPC:DPPA bilayers. This is un-

doubtedly due to the larger chain tilt for the bilayers containing DPPC compared to the bilayers containing DPPE.5 The wide-angle diffraction data, the reciprocal space analysis, and the electron density profiles all show that the packing of the hydrocarbon chains and structure of the bilayer were not appreciably changed by the application of osmotic pressures up to 3.2 × 107 dyn/cm2. Similar observations have previously been made for a variety of lipid bilayer systems, including egg phosphatidylcholine (EPC),42 DPPC,42 EPC:cholesterol,34 sphingomyelin,61 and EPC:ganglioside GM1.62 Interbilayer Pressures. The observed invariance of bilayer structure indicates that most of the water removed (61) McIntosh, T. J.; Simon, S. A.; Needham, D.; Huang, C.-h. Biochemistry 1992, 31, 2020. (62) McIntosh, T. J.; Simon, S. A. Biochemistry 1994, 33, 10477.

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Figure 5. The logarithm of applied pressure (log P) plotted versus the lamellar repeat period (d) minus the peak-to-peak distance in the electron density profiles (dp) for DPPE (open circles), 19:1 DPPE:DPPA (open squares), 4:1 DPPE:DPPA (open diamonds), DPPC (solid circles), 19:1 DPPC:DPPA (solid squares), and 4:1 DPPE:DPPA (solid diamonds). The dotted and solid lines represent the calculated electrostatic pressure for 19:1 DPPC:DPPA and 4:1 DPPC:DPPA bilayers (see text for details).

by this range of osmotic pressure was being removed from the fluid space between bilayers and not from within the bilayer structure. This implies that most of the applied osmotic stress was balanced by interbilayer (rather than intrabilayer) pressures.41 For gel phase bilayers where the undulation38,63 and protrusion pressures35 are both small,45,46 the total pressure P between bilayers can be expressed as

P ) Psr + Ph + Pes - Pv

(2)

where Psr represents a very short-range steric repulsion between headgroups from apposing bilayers,33,34 Ph represents the short-range repulsive hydration pressure,25,31,32,42 Pes is the repulsive electrostatic pressure, and Pv is the attractive van der Waals pressure. The shortrange steric repulsion (Psr) between headgroups from apposing bilayers should not be a factor in these experiments since for PC bilayers it is prominent only for log P > 8.33,34,45 We now examine the observed pressuredistance relations (Figure 4) in terms of the other component pressures given in eq 2, first considering the electrostatic pressure. With the assumptions of a constant potential and no counterion binding, the repulsive electrostatic pressure between two planar charged surfaces in a 1:1 electrolyte can be expressed as

Pes ) 64kTFγ2 exp(-KD)

(3)

where k is the Boltzmann constant, T is temperature, F is the bulk ionic concentration, γ ) tanh(eψ/4kT) where e is the electronic charge and ψ is the bilayer surface potential, 1/K is the Debye length which is 9.6 Å for 100 mM NaCl, and D is the distance between the charged layers.64 The surface potential can be calculated from the Gouy equation

sinh(eψ/2kT) ) σ/(80kTF)1/2

(4)

(63) Helfrich, W.; Servuss, R.-M. Il Nuovo Cimento 1984, 3, 137.

where σ is the surface charge density,  is the dielectric constant, and 0 is the permittivity of free space.65 For D ) 0, we use the position of the phosphate groups as estimated from the position of the headgroup peak in the electron density profiles and the data of Hitchcock et al.3 that show that the center of the phosphate group is about 1 Å out toward the fluid space from the center of the electron density peak in comparable resolution electron density maps. Thus, we use the plane of the phosphate groups as the plane origin in eq 3 and set D ) d - (dpp + 2 Å). To calculate σ, we assume that the area per lipid molecule in the DPPC bilayer is 48 Å2 9 and that each DPPA molecule is singly charged. This latter assumption can be justified since these experiments were performed at pH 7.0 and, in 0.1 M ionic strength, the two pKa’s of DPPA are 3.0-4.0 and 8.0-9.0.66 Using these procedures we calculated Pes for 19:1 DPPC:DPPA and 4:1 DPPC: DPPA (dotted and solid lines in Figure 5). For DPPE, the only parameter that is expected to be different in the calculations is the area per lipid molecule, which on the basis of the wide-angle diffraction data should be about 40 Å2 for DPPE and DPPE:DPPA bilayers. Thus, the electrostatic pressures for 19:1 DPPE:DPPA and 4:1 DPPE:DPPA would be predicted to be slightly larger in magnitude than the pressures shown by the dotted and solid lines in Figure 5. For values of d-dpp greater than about 20 Å, the calculated electrostatic pressure (solid line) approximated quite closely the data points for both 4:1 DPPE:DPPA and 4:1 DPPC:DPPA bilayers. This good agreement suggests that the assumptions involved in eqs 3 and 4 were reasonable. For values of d-dpp less than about 20 Å, the data points for 4:1 DPPC:DPPA deviated upward and away from the solid line and superimposed with the data points for DPPC, and for values of d-dpp less than about 15 Å the data points for 4:1 DPPE:DPPA superimposed with (64) Israelachvili, J. N. Intermolecular and Surface Forces; Academic Press: London, 1985. (65) McLaughlin, S. Ann. Rev. Biophys. Biophys. Chem. 1989, 18, 113. (66) Tatulian, S. A. In Phospholipids Handbook; Cevc, G., Eds.; Marcel Dekker, Inc.: New York, 1993.

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the data points for DPPE. For 19:1 DPPC:DPPA, for values of d-dpp greater than about 20 Å, the theoretical values of the calculated electrostatic pressure (dotted line) fell below the experimental data points. A possibility to explain this mismatch is that, with a lower charge density, DPPC:DPPA bilayers act as if they had, on average, more than one charge per DPPA. If the buffering at the bilayer surface were not complete, the higher the surface charge density the lower the pH. This means that bilayers containing lower concentrations of DPPA could be at a higher pH, close to pH 7, and near the range of the higher pKa of the phosphate (8.0-9.0).66 Thus, to a good approximation, for d-dpp > 20 Å, the pressure-distance relations for 4:1 DPPC:DPPA, 4:1 DPPE:DPPA, and 19:1 DPPC:DPPA can be understood in terms of the electrostatic pressure (solid and dotted lines, Figure 5). However, several interrelated aspects of the pressure-distance relations can not be explained simply in terms of Pes, including the presence and position of the sharp upward breaks in the pressure-distance curves (at d-dpp ≈ 20 Å for 4:1 DPPC:DPPA and at d-dpp ≈ 15 Å for 4:1 DPPE:DPPA), the observation that, unlike 19:1 DPPC:DPPA bilayers, 19:1 DPPE:DPPA bilayers do not disjoin due to Pes, and the difference in the pressuredistance relations for uncharged DPPE and DPPC. We first consider the reason for the sharp upward breaks in the pressure-distance curves for 4:1 DPPC:DPPA, 19:1 DPPC:DPPA, and 4:1 DPPE:DPPA. There are two possible reasons to explain these upward breaks: (1) doublelayer theory (eq 3) is only valid for surface separations greater than about 1 Debye length and, with a constant charge density, upward breaks can occur in Pes at short spacings,64 and (2) at short spacings the hydration pressure becomes the dominant pressure (Ph > Pes).58-60 We argue that the second possibility is the more important factor for two reasons. First, the upward breaks occur at different values of d-dpp for 4:1 DPPE:DPPA and 4:1 DPPC:DPPA. Since the charged group should be at the same location in the two types of bilayers (at D ) 0 as defined above), the difference in position of the upward break can not be explained solely by electrostatic pressure. Second, for both DPPE:DPPA (Figure 4A) and DPPC: DPPA (Figure 4B), the pressure-distance data superimpose with the uncharged DPPE and DPPC data for small values of d-dpp. For uncharged DPPE and DPPA the major repulsive pressure present is Ph.25,32,41,42,67 Thus, as apposing DPPE:DPPA and DPPC:DPPA bilayers are squeezed together against Pes, they eventually run into the much stronger short-range hydration pressure (Ph), causing the observed upward breaks in the pressuredistance relations. We also note that at small fluids spaces, where the concentration of the counterion (Na+) is high, the process of charge neutralization could occur with the counterion binding to and neutralizing DPPA-.68 We next consider possible reasons for the related observations that 19:1 DPPE:DPPA bilayers do not disjoin due to Pes and the pressure-distance relations are different for uncharged DPPE and DPPC. As noted in the Introduction, the observation that DPPE bilayers do not take up as much water as do DPPC bilayers can be explained by DPPE bilayers having either smaller repulsive pressure(s) or larger attractive pressure(s). The following calculations of the adhesion energy for DPPE provide strong evidence that the latter possibility must be true. The adhesion energy between PE bilayers can be estimated using the pressure-distance data and double-layer theory. (67) McIntosh, T. J.; Magid, A. D.; Simon, S. A. Biophys. J. 1989, 55, 897. (68) Chan, D. C.; Pashley, R. M.; White, L. R. J. Colloid Interface Sci. 1980, 77, 283.

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Our results (Figure 1A) indicate that the introduction of about 10 mol % DPPA adds just enough charge to disjoin at least some of the DPPE bilayers. Therefore, we use the electrostatic pressure for 9:1 DPPE:DPPA, calculated from eq 3, as a measure of the minimum repulsive pressure necessary to disjoin DPPE bilayers. By integration of eq 3 from D ) ∞ to 13.2 Å (the value of D at the equilibrium fluid separation for DPPE), we obtain the adhesion energy per unit area of E ≈ -0.7 erg/cm2. This value can be compared to values for gel phase DPPE and DPPC of E ) -0.80 and -0.15 erg/cm2, respectively, obtained with the surface force apparatus.27 Although there is good agreement in the values of adhesion energy (E) for DPPE using these two very different methods, the agreement may be somewhat fortuitous given the various approximations made in our calculations and that the adhesion energy obtained with the surface force apparatus may contain an additional attraction due to the interaction between the mica surfaces supporting the bilayers.69 However, for the purpose of this paper, the critical point is that for gel phase bilayers the adhesion energy is considerably larger for PE than for PC bilayers. It should also be noted that for liquid crystalline phase bilayers, both the osmotic stress technique25 and the pipet aspiration method43 indicate that the adhesion energy is larger for PE than for PC bilayers. The adhesion energy (E) between bilayers can be written

E ) Ea + Er

(5)

where Ea and Er represent the total attractive and repulsive energies, respectively, at the equilibrium fluid separation. The attractive energy due to the van der Waals pressure can be expressed as

Ev ) -(H/12π)(1/dv)2

(6)

where H is the Hamaker constant and dv represents the distance from the plane of origin of the van der Waals pressure. Given the similarity in composition of DPPE and DPPC and on the basis of theoretical treatments of the van der Waals pressure,70 we assume that the Hamaker constant should be approximately the same for DPPE and DPPC bilayers and set H ) 3 × 10-14 erg.71 We also assume that the plane of origin for Ev is at the center of the DPPE headgroup region, near the center of mass, so that dv ) d - dpp. With these assumptions we calculate that at the equilibrium fluid spacing Ev ≈ -0.03 erg/cm2. Thus, for DPPE bilayers our estimated adhesion energy (E ) -0.7 erg/cm2) is much larger in magnitude than the van der Waals energy calculated with this standard formalism. Since the repulsive component of the energy (Er) must be positive, the only way that eq 5 can be balanced for DPPE is for there to be an additional attractive pressure besides the usual representation of the van der Waals pressure. In contrast, for DPPC it has previously been shown that eq 5 can be balanced by considering only the repulsive hydration pressure and the attractive van der Waals pressure.45 Attractive interactions that have been proposed for PE bilayers involve interbilayer interactions between the N+H3 group in one bilayer and the PO4- group in the apposing bilayers, through either electrostatic interactions,7,12,39 direct hydrogen bonding,12 or hydrogen-bonded water bridges.7,24 Such short-range attractive interactions are consistent with the X-ray data (Figure 4A) which show (69) Parsegian, V. A. Langmuir 1993, 9, 3625. (70) Nir, S. Prog. Surf. Sci. 1976, 8, 1. (71) Needham, D.; Haydon, D. A. Biophys. J. 1983, 41, 251.

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a discontinuous disjoining of PE bilayers with increasing DPPA concentration. These types of interactions are more probable for PE bilayers than PC bilayers for the following reasons. In terms of direct electrostatic interactions, the lipid headgroups have somewhat less motional freedom in hydrated PE bilayers than in hydrated PC bilayers,16,72 and since the trimethylammonium moiety of PC has a greater volume than the amine of PE, the positive charge of the PE headgroup is more localized in the plane of the bilayer. As shown in Figure 4A, the average distance between the apposing headgroups in fully hydrated DPPE is about 7 Å. Taking into account the thermal motion of the DPPE headgroup16 which could extend the amine group about 2 Å further into the fluid space, we estiamte that in fully hydrated DPPE bilayers the closest approach of the outer edges of the amine and phosphate moieties from apposing bilayers would be about 3 Å. Thus, salt bridge formation or direct H-bonding between the N+H3 group in one bilayer and the PO4- group in the apposing bilayer seems, on average, unlikely. However, since the size of a water molecule is about 3 Å and the typical H-bond distance is over 1.5 Å,64 the distance between these groups could be spanned by only one or two water molecules hydrogen bonded to the N+H3 and PO4- groups from apposing bilayers. In terms of hydrogen bonding, recent molecular dynamics simulations show that water molecules are hydrogen bonded to PE headgroups,39,73 whereas water forms a clathrate-like structure around the PC headgroup.39,74 To account for the adhesion energy for DPPE (E ) -0.7 erg/cm2), it is instructive to compare this value with that of thermal energy (kT ) 4.1 × 10-14 erg/molecule). Since the molecular area of DPPE is 40 Å2, then the magnitude of E ≈ 0.07kT. This can be compared to a direct Coulombic interaction between N+H3 group in one bilayer and the PO4- on the apposing bilayer which could produce an attractive interaction energy of about 100-200kT and a hydrogen bond which has a magnitude of about 4-15kT. Thus, although from first principles one cannot calculate the small free energy between PE bilayers because of the difficulty in accounting for the numerous enthalpic and entropic contributions, it appears that a small fraction of direct electrostatic interbilayer interactions or indirect hydrogen-bonded water interactions between the N+H3 group in one bilayer and the PO4- group in the apposing bilayers could account for the additional attractive interactions in PE bilayers. In support of the idea of these interbilayer attractive interactions is the observation that the Lβ gel phase in PE bilayers is metastable and will, given sufficient time, collapse to a less-hydrated crystalline state.11,75,76 The time to form this crystalline phase, usually on the order (72) Michaelson, D. M.; Horowitz, A. F.; Klein, M. P. Biochemistry 1974, 13, 2605. (73) Zhou, F.; Schulten, K. J. Phys. Chem. 1995, 99, 2194. (74) Essmann, U.; Perera, L.; Berkowitz, M. L. Langmuir, in press, 1995.

McIntosh and Simon

of days, is much longer then the time needed to perform osmotic stress experiments and is probably related to the time necessary for the P--N+ dipoles in one bilayer to align fully with the oppositely oriented dipoles in the apposing monolayer to form direct N+H3-PO4- interactions. These attractive interactions have sufficient energy to eliminate the polarized interlamellar waters that could not be eliminated by osmotic stresses up to 3 × 107 dyn/ cm2 (Figure 4A). The direct interactions between PO4and N+H3 groups in adjacent molecules in the same DPPE monolayer diminish water penetration in the polar headgroup region. To form the metastable Lβ phase, work has to be done on the system by first incubating the lipid above its gel to liquid crystalline phase transition (see Materials and Methods). Once cooled to the Lβ phase, the waters of hydration are “kinetically trapped” until the randomly arranged DPPE domains between apposing monolayers become aligned to a sufficient extent to squeeze the entropically restricted interbilayer water molecules into the bulk phase. Since we estimate that the closest approach of the outer edges of the amine and phosphate moieties from apposing bilayers would be only about 3 Å, distance fluctuations can form a few of the PO4--N+H3 interactions that can nucleate and grow. Given that the lipids are in the gel state where their lateral diffusion constant is low, this nucleation process would be expected to be slow. Another difference between PC and PE bilayers is that the fluid separation in fully hydrated bilayers is the same for gel and liquid crystalline phase PEs,7 whereas the equilibrium fluid separation is considerably larger for the liquid crystalline phase compared to that of the gel phase for PC bilayers.28,42 This means that the difference in fluid spacing between hydrated liquid crystalline PE and PC42 is even larger than the difference between gel phase DPPE and DPPC. Liquid crystalline PC bilayers swell to a greater extent than gel phase PC bilayers primarily due to the presence of the repulsive undulation pressure in liquid crystalline bilayers.28,38,67 Since the area compressibility moduli are similar for liquid crystalline PC and PE bilayers,43 and the bilayer thicknesses are comparable,7,42 the bending moduli and repulsive undulation pressures should be comparable for liquid crystalline PC and PE bilayers. However, in the case of PE bilayers, we argue that attractive interactions, probably due to H-bond water bridges or electrostatic interactions, overcome the undulation pressure and prevent liquid crystalline PE bilayers from swelling. Acknowledgment. We thank Drs. Klaus Gawrisch and Max Berkowitz for many helpful comments. This work was supported by Grant GM-27278 from the National Institutes of Health. LA950833G (75) Chang, H.; Epand, R. M. Biochim. Biophys. Acta 1983, 728, 319. (76) Wilkinson, D. A.; Nagle, J. F. Biochemistry 1984, 23, 1538.